JP6075087B2 - Fluorometer inspection method, inspection reagent container, and fluorimeter - Google Patents

Description

  The present invention relates to a fluorescence measurement technique.

As one field of light measurement, a fluorescence measurement technique for measuring fluorescence emitted from a substance is known. Material analysis by fluorescence measurement (fluorescence analysis method) is characterized by high sensitivity and high selectivity compared to absorptiometry and the like, and is effective when performing identification or quantification of a sample.
Sample identification and quantification by fluorescence measurement is limited to the case where the target substance is a fluorescent substance. However, in recent years, fluorescent labeling methods for labeling target substances with reagents (fluorescent reagents) made of fluorescent dyes have been developed, and fluorescent reagents are commercially available for various substances. For this reason, identification and quantification of various target substances by fluorescence measurement has become possible, and applications are being studied in many fields such as research and development of new drugs and new materials, process monitoring in plants, and environmental evaluation.

  In such fluorescence measurement, the measurement may be performed a plurality of times, and the ratio of the measurement value (fluorescence intensity) in each measurement may be used as a measurement result. For example, Patent Document 1 discloses a technique for identifying and quantifying a sample by fluorescence measurement using an immune reaction. This technology uses the fact that quenching (fluorescence quenching) that has occurred in fluorescent dyes is eliminated by an immune reaction, and is a technology for identifying and quantifying samples using the increase in fluorescence intensity before and after the reaction as an index. is there.

Japanese Patent Laid-Open No. 10-19892

Like other light measurements, it is inevitable that some noise is included in the measurement result in the fluorescence measurement. In order to perform highly accurate measurement, it is necessary to sufficiently remove the noise. In this specification, noise has a broad meaning as a factor that causes a decrease in measurement accuracy.
When performing the measurement twice as described above, a lot of noise can be removed by taking a ratio. For example, the noise generated when the output of the light source (excitation light) that irradiates the sample to generate fluorescence fluctuates for some reason, shortens the measurement interval to the extent that it is not affected by such fluctuation. It can be removed by measuring twice or dividing the light from the light source, obtaining reference data from one light, and taking the ratio with the measurement data obtained by the other light.
However, according to the research of the inventors, it has been found that in fluorescence measurement, there is noise that cannot be removed only by taking a ratio, and some of the noise is due to circumstances specific to fluorescence measurement. The invention of the present application is based on this finding, and has been made to solve the problem of noise peculiar to fluorescence measurement that cannot be removed only by taking a ratio.

In order to solve the above-mentioned problem, the invention according to claim 1 of the present application is arranged such that a sample container in which a liquid phase sample is accommodated in an accommodating portion is disposed so that the accommodating portion is a measurement position, and excitation light is irradiated to the measurement position. A method for checking a fluorometer that captures fluorescence generated in a liquid phase sample with a detector,
It is an inspection method using an inspection reagent container in which the first and second two liquid phase reagents for inspection whose fluorescence intensity ratio is known can be sequentially arranged at the measurement position,
The first measurement in which the detection light is captured by the detector by irradiating the measurement light with the excitation light from the light source in a state where the storage portion of the inspection reagent container containing the first inspection liquid phase reagent is positioned at the measurement position Steps,
Second measurement in which the measurement light is irradiated with the excitation light from the light source and the generated fluorescence is captured by the detector in a state where the container of the inspection reagent container containing the second inspection liquid phase reagent is positioned at the measurement position. Steps,
The ratio of the fluorescence intensity captured by the detector in the first measurement step and the fluorescence intensity captured by the detector in the second measurement step is obtained, and the obtained ratio and the known fluorescence intensity And a calculation step of calculating a difference from the ratio.
In order to solve the above-mentioned problem, the invention according to claim 2 is the configuration of claim 1, wherein the second inspection liquid phase reagent is made of a different material from the first inspection liquid phase reagent. It consists of a mixture,
After the first measurement step, another material is mixed with the first inspection liquid phase reagent stored in the storage unit, and the second inspection liquid phase reagent is mixed in the storage unit. It has a configuration in which the second measurement step is performed after that.
In order to solve the above problem, the invention according to claim 3 is the configuration according to claim 2 , wherein the first inspection liquid phase reagent and the other material are solutions of the same fluorescent reagent in different concentrations. It has a structure that it is formed by dissolving.

In order to solve the above-mentioned problem, the invention described in claim 4 is the reagent container used in the inspection method of the fluorometer according to claim 1, 2, or 3, wherein the known fluorescence intensity is used. It has a display that displays the ratio.
Further, in order to solve the above-mentioned problem, the invention according to claim 5 is the configuration according to claim 4, wherein the display unit displays a determination value in addition to the known fluorescence intensity ratio. The judgment value is a reference value for the magnitude of the difference between the obtained ratio and the known fluorescence intensity ratio, and when the difference exceeds the possibility judgment value, the fluorometer is used. The configuration is such that the value is disabled.
In order to solve the above problem, the invention according to claim 6 is the code display unit according to claim 4 or 5, wherein the display unit can be read by a code reader included in the fluorometer. It has the structure of being.
In order to solve the above-mentioned problem, the invention described in claim 7 is an inspection reagent container used in the inspection method of the fluorometer according to claim 2, wherein the first inspection liquid phase reagent is used. And a second accommodating part accommodating the other material, and moving the another material from the second accommodating part to the first accommodating part, It has a structure that can be mixed with the first liquid phase reagent.

In order to solve the above problem, the invention according to claim 8 is a fluorometer in which the fluorometer inspection method according to claim 1, 2, or 3 is implemented,
A storage unit, an arithmetic processing unit, and a display;
The storage unit measures the fluorescence intensity of the first inspection liquid phase reagent measured in the first measurement step, and the second inspection liquid phase reagent measured in the second measurement step. The measured value of the fluorescence intensity of
An inspection program is stored in the storage unit and can be executed by the arithmetic processing unit.
The inspection program has a configuration in which the ratio of the fluorescence intensity of the first inspection liquid phase reagent and the fluorescence intensity of the second inspection liquid phase reagent is obtained and displayed on the display.
In order to solve the above problems, the invention according to claim 9 is a fluorometer in which the fluorometer inspection method according to claim 1, 2, or 3 is implemented,
A storage unit, an arithmetic processing unit, and a display;
The storage unit measures the fluorescence intensity of the first inspection liquid phase reagent measured in the first measurement step, and the second inspection liquid phase reagent measured in the second measurement step. The measured value of the fluorescence intensity of
An inspection program is stored in the storage unit and can be executed by the arithmetic processing unit.
The inspection program obtains the ratio between the fluorescence intensity of the first inspection liquid phase reagent and the fluorescence intensity of the second inspection liquid phase reagent, and then whether or not the obtained difference exceeds the determination value. When it is judged and exceeded, the program is a program for displaying on the display that the fluorometer is unusable.

As described below, according to the invention described in claim 1 of the present application, two liquid phase reagents for which the generated fluorescence intensity ratio is known are put into the fluorometer to be inspected, and measurement is performed. Since the ratio of the values is obtained, it is possible to check how much the obtained ratio is dissociated from the standard fluorescence ratio. For this reason, it is possible to prevent an erroneous measurement result from being obtained by performing measurement without knowing the decrease in measurement accuracy. In addition, a method optimized as an inspection method for a fluorometer that obtains the ratio of measured values of two samples as a measurement result is provided, and factors such as light output fluctuation and light shielding due to contamination of the optical system are provided. It becomes possible to easily check the influence of background noise while canceling.
According to the second or seventh aspect of the invention, in addition to the above effect, the second measurement step can be performed subsequent to the first measurement step without changing the position or posture of the reagent container. For this reason, an inspection without error can be easily performed.
According to the invention of claim 3, in addition to the above effect, the first inspection liquid phase reagent and the second inspection liquid phase reagent are the same reagent and have different concentrations. Inspection without any problems can be easily performed.
According to the invention described in claim 4, in addition to the above-described effect, the inspection reagent container has a display unit displaying a known fluorescence ratio, so whether or not the fluorometer can be used. Can be easily determined.
Further, according to the invention described in claim 5, in addition to the above effect, the display unit also displays a determination value of whether or not the fluorometer is usable in addition to the known fluorescence ratio, so it is easy to determine whether or not the fluorometer can be used. Can be done without error.
According to the invention described in claim 6, in addition to the above-described effect, the display unit is a code display unit that can be read by a code reader included in the fluorometer. Is suitable for providing digital information to a fluorometer.
According to the invention described in claim 8 or 9, in addition to the above-described effect, the calculation for obtaining the fluorescence intensity ratio from the measured value and the calculation for obtaining the difference by comparing the obtained fluorescence intensity ratio with the standard fluorescence intensity ratio are performed. In this respect, the fluorometer is highly convenient.

It is a schematic perspective view of an example of a fluorometer that is inspected by the method of the embodiment. FIG. 2 is a schematic front sectional view of the fluorometer shown in FIG. 1. It is the figure which showed an example of the sample container used for the fluorimeter shown in FIG.1 and FIG.2, (1) is an external appearance schematic diagram, (2) is a front cross-sectional schematic diagram. It is the block diagram shown about the signal processing system of the fluorometer shown in FIG.1 and FIG.2. It is a figure which shows the result of the experiment which confirmed that the measurement precision fall by a foreign material penetration | invasion cannot be removed only by taking the ratio of a measured value. It is the schematic of the inspection reagent container which concerns on embodiment of this invention. It is the figure which showed schematic structure and the operation principle of the barcode reader 7 shown in FIG. It is the schematic shown about the example of the format of the code display part 96 shown in FIG. It is the flowchart which showed the outline of the inspection program.

Hereinafter, modes (embodiments) for carrying out the present invention will be described.
The method of the embodiment is a method for inspecting a fluorometer, and in particular, a method for inspecting whether or not the measurement accuracy lower than the limit can be caused in the fluorometer. First, an example of a fluorometer to be inspected by the method of the embodiment will be described with reference to FIG. FIG. 1 is a schematic perspective view of an example of a fluorometer to be inspected by the method of the embodiment, and FIG. 2 is a schematic front sectional view of the fluorometer shown in FIG. The following description of the fluorometer also serves as the description of the fluorometer of the embodiment.

The fluorometer 1 shown in FIGS. 1 and 2 is assumed to be measured at a site where a sample is collected or at a location close thereto. That is, it is not always installed in a special room such as a measurement room or a laboratory, but is a portable fluorometer. The fluorometer 1 measures the intensity of fluorescence generated in a liquid phase sample (hereinafter referred to as a liquid phase sample), and is a container (hereinafter referred to as a sample) for holding the liquid phase sample at a measurement position. Container).
More specifically, as shown in FIG. 1, the fluorometer 1 is a flat, substantially rectangular parallelepiped box-like thing as a whole. Since it is portable, its size is about the size of a person's palm or slightly larger.

An opening 11 is formed on the upper surface of a flat, substantially rectangular parallelepiped box-shaped casing 10, and an opening / closing lid 12 is provided in the opening 11. A container holding part 5 is provided in the casing 10, and when the opening / closing lid 12 is opened, the insertion hole 50 at the upper end of the container holder 5 is exposed. A sample container (not shown) in FIG. 1 is inserted into the container holder 5 through the insertion hole 50 and held in the container holder 5 so that the sample container is mounted at a predetermined position in the casing 10. In addition, on the front surface of the casing 10, various operation buttons 141 to 146 including a display 13 for displaying information necessary for measurement and measurement results, and measurement buttons 141 are provided.
As shown in FIG. 2, in the casing 10, a light source 2 that emits light (excitation light) having a wavelength capable of exciting a sample to emit fluorescence, a detector 3 that captures the generated fluorescence, and excitation An optical system 4 that guides light to the sample and guides the generated fluorescence to the detector 3, and a container holder 5 that holds the sample container 8 so that the liquid phase sample is positioned at the excitation light irradiation position (measurement position). Is provided.

FIG. 3 is a view showing an example of a sample container used in the fluorometer shown in FIGS. 1 and 2, wherein (1) is a schematic external view, and (2) is a schematic front sectional view.
Although the sample itself may be in a liquid phase, it is assumed that the sample is in a solid state such as a powder, and the generated fluorescence is measured after dissolving in a solution and making it into a liquid phase. It has become. For this reason, the sample container 8 also serves as a purpose of providing a solution for dissolving the sample. Further, the fluorometer 1 of this example is for performing fluorescence measurement utilizing an immune reaction, and the sample container 8 is one in which two solutions are stored in advance.

  Specifically, the sample container 8 has a storage part (hereinafter referred to as a first storage part) 81 positioned at the measurement position and a second storage part 82 different from this. A solution (hereinafter referred to as an antibody solution) 83 in which an antibody is dissolved is previously stored in the first storage portion 81. The second accommodating portion 82 is partitioned by a partition wall 84 that can be broken with respect to the first accommodating portion 81. Here, the sample is dissolved and adjusted to a predetermined concentration and then charged into the antibody solution 83. A solution (hereinafter referred to as an adjustment solution) 85 is stored.

In addition, as shown in FIG. 3, the sample container 8 is elongate. The container holding part 5 is a frame-shaped member that matches the size and shape of the sample container 8. As shown in FIG. 3, the 1st accommodating part 81 is provided in the lower end part of the sample container 8, and the 2nd accommodating part 82 is provided in the middle part. The partition wall 84 serves as the bottom wall of the second accommodating portion 82.
The upper end of the sample container 8 is an opening, and a cap-shaped lid 86 is provided in this opening. When the sample is loaded, the lid 86 is opened. In addition, when the sample container 8 is correctly held and attached to the container attachment portion 5, the first storage portion 81 is located at the measurement position. The measurement position is a position where excitation light is irradiated through the optical system 4 and generated fluorescence is captured by the detector 3 through the optical system 4.

In the fluorometer 1 shown in FIG. 2, an LED lamp is used for the light source 2 provided in the casing 10 in consideration of cost advantage and power saving. For example, one that emits green light having a wavelength of 525 nm and that has an output of about 2 mW is used.
The optical system 4 includes a condenser lens 41 that condenses light from the light source 2, a dichroic mirror 42 for bending the optical path and selecting light, and filters 43 and 44 disposed on the optical path. The The light source 2 is configured to emit light downward, and the dichroic mirror 42 is disposed at an oblique angle of 45 ° below the light source 2. The dichroic mirror 42 reflects light having the excitation light wavelength and transmits light having the fluorescence wavelength to be measured.

The detector 3 is disposed at a position opposite to the container mounting portion 5 with the dichroic mirror 42 interposed therebetween. As the detector 3, for example, a detector that performs photoelectric conversion using a silicon photodiode is used.
An excitation light filter 43 is disposed between the light source 2 and the dichroic mirror 42, and a fluorescence filter 44 is disposed between the dichroic mirror 42 and the detector. When green light of 525 nm is used as excitation light, a filter that transmits light in the wavelength range of about 510 to 545 nm and reflects light in other wavelength ranges is used as the excitation light filter 43. In this case, the fluorescence wavelength to be measured is about 550 to 630 nm, and the filter 44 for fluorescence transmits light in the wavelength range of about 570 to 610 nm and reflects light in other wavelength ranges. The The condenser lens 41 irradiates the liquid phase sample in the accommodating portion 81 with the light from the light source 2 as a thin beam, and collects the fluorescence emitted from the liquid phase sample and makes it incident on the detector 3. is there.

Next, the signal processing system of the fluorometer shown in FIGS. 1 and 2 will be described. FIG. 4 is a block diagram showing a signal processing system of the fluorometer shown in FIGS. 1 and 2.
As shown in FIG. 2, a control box 60 is provided in the casing 10. In the control box 60, there is provided a control unit 6 that controls each part and performs signal processing. The control unit 6 includes a processor 61 that executes various programs, a memory 62 that stores data and programs, and the like.

The detector 3 is a photoelectric conversion unit (silicon photodiode in this example) 31 that receives fluorescence, an amplifier 32 that amplifies the output signal of the photoelectric conversion unit 31, and a fluorescence intensity signal that is output based on the amplified signal. Output circuit 33. The output circuit 33 includes a calibration circuit for displaying the light intensity (fluorescence intensity) as an absolute value as necessary.
In addition to the output from the detector 3, operation signals from the operation buttons 141 to 146 and signals from the power switch are input to the control unit 6. In addition, a display 13 is connected to the control unit 6 via an interface (not shown).
As shown in FIG. 2, a battery case 600 is provided in the casing 10. A battery that supplies necessary voltages to the light source 2, the detector 3, the control unit 6, and the like is mounted on the battery case 600.

  When measurement is performed using such a fluorometer 1, a predetermined amount of sample is collected, put into the second container 84, and dissolved in the adjustment solution 85. In this state, the sample container 8 is mounted on the container mounting portion 5 and the light source 2 is turned on to perform measurement. In this state, the sample is only in the second container 82, and the antibody solution 83 is only in the first container 81 at the measurement position. Therefore, the generated fluorescence is measured by irradiating the antibody solution 83 in a state where the sample is not loaded with excitation light. Thereafter, the partition wall 84 of the sample container 8 is broken using a jig (not shown), the adjustment solution 85 in which the sample is dissolved is put into the antibody solution 82 and mixed, and then the measurement is performed again. . If the sample is an antigen, an immune reaction occurs, quenching is eliminated, and fluorescence generated in the antibody solution 83 is enhanced. Therefore, by calculating the ratio of the two measured values, it is possible to identify whether the sample is an antigen. Further, the sample can be quantified by comparing the fluorescence enhancement ratio with predetermined calibration data.

  The program stored in the memory 62 includes a program for the measurement. Specifically, a program that displays on the display 13 a display that prompts the user to mount the sample container 8 or displays a display that prompts the user to press the measurement button (button that turns on the light source 2) 141 after the mounting is completed. In addition, it is a program or the like that stores the measurement result of each time in the memory 62 and calculates the ratio to obtain the measurement result.

In fluorescence measurement using such a fluorometer 1, it is inevitable that noise is included in the measurement result as described above. The noise is generated when the light source 2 is deteriorated and the output is reduced, or dirt is attached to the optical system 4 to partially block the excitation light or generated fluorescence. These noises can be removed by taking a ratio of measured values.
For example, a standard fluorescent reagent whose fluorescence emission intensity is known is used, dissolved in a solution at a predetermined concentration, and stored in the first storage portion of the sample container, and the measurement is performed. The result is used as the reference value Vc. Next, another sample container in which the sample is dissolved is replaced and attached to the solution in the first container (or the container in the first container is replaced with the sample solution), and measurement is performed. By taking the ratio of the measured value V obtained in this way to Vc, the measurement can be performed without being affected by noise caused by the deterioration of the light source 2 over time, the contamination of the optical system 4, and the like.

However, according to the inventor's research, it has been found that there is noise that cannot be removed only by taking a ratio of measured values, and that noise is due to factors specific to fluorescence measurement. Hereinafter, this point will be described in detail.
In the fluorescence measurement research conducted by the inventor, even if noise is removed by taking the ratio of measured values, fluctuations in measurement results that cannot be ignored occur, and this may cause a problem that the measurement accuracy cannot be increased. found. The inventor conducted further diligent research on the cause of this problem. This is due to the influence of foreign matter such as dust and dust that has entered the optical system 4, and in particular, the foreign matter that is a fluorescent material enters the optical system 4. It has become clear that
The foreign material that can enter the casing 10 of the fluorometer is typically dust or dirt. Some dust and dust are fluorescent materials. For example, lint fragments from clothes and the like often become dust or dust, but lint may be a fluorescent material. This is the case where the fiber is a fluorescent material, dyed with a fluorescent paint, or a detergent containing the fluorescent material remains. In addition, human sebum causes dust and dust, but sebum also contains fluorescent components.

These foreign substances, which are fluorescent substances, enter the casing 10 and adhere to the elements of the optical system 4 when the opening / closing lid 12 is opened for mounting the sample container 8. For example, it adheres to the surface of the condenser lens 41. Noise due to the blocking of excitation light and fluorescence by foreign substances can be removed by taking the ratio of measured values as described above. Even if the amount of foreign matter fluctuates in an unstable manner, this noise can be measured by making two measurements consecutively in time (measured so that there is no change in the amount of foreign matter between the two measurements). If done, it will not affect the measurement accuracy. However, when the foreign substance is a fluorescent substance and the wavelength of the generated fluorescence overlaps the range of the measurement wavelength (when it passes through the fluorescent filter 44), the fluorescence from the foreign substance appears in the output from the detector 3. It will be included.
The noise when the foreign matter that has entered the optical system 4 is a fluorescent material is the same type as the background noise, and cannot be removed simply by taking a ratio. Therefore, it is not possible to prevent this kind of decrease in measurement accuracy simply by taking two measurements and taking the ratio.

FIG. 5 is a diagram showing a result of an experiment confirming that such a decrease in measurement accuracy due to the entry of a foreign substance cannot be removed only by taking a ratio of measured values. In the experiment whose results are shown in FIG. 5, the measurement was performed using the same fluorometer and using a container having the same structure as the sample container 8 described above as a reagent container. Two different fluorescent liquid phase reagents a and b were sequentially accommodated in the first accommodating portion of the reagent container, and the fluorescence intensity was measured for each. Specifically, TAMRA was used as a fluorescent reagent, and a solution obtained by dissolving this in a solution at different concentrations was prepared as a liquid phase reagent. One is 1 nmol / liter (liquid phase reagent a) and the other is 5 nmol / liter (liquid phase reagent b). In practice, TAMRA is dissolved in a PBS solution (phosphate buffered saline solution) to a concentration of 1 nmol / liter (liquid phase reagent a), and this is stored in the first storage portion 81 in an amount of 75 μL. The first measurement was performed. Next, the liquid phase reagent in the first container was left as it was, and 25 μL of TAMRA dissolved in a PBS solution to a concentration of 17 nmol / L was added and mixed therewith. Thereby, the liquid phase reagent b was prepared in the 1st accommodating part, and the 2nd measurement was performed in this state.
In the experiment, in order to grasp the amount of background noise, the light source 2 was turned on with no reagent container mounted, the output of the detector 3 was confirmed, and the value was used as the offset value.

  Further, in the experiment, in order to grasp the influence of foreign matter contamination, dust was intentionally introduced into the casing 10 from the insertion hole 11 as a foreign matter, and measurement was performed. First, the above-mentioned measurement (measurement with respect to the liquid phase reagents a and b) was performed (first measurement set) in a state where dust was not added. Next, after putting a small amount of dust through the insertion hole 11, the same measurement was performed twice (second measurement set). Furthermore, after adding dust, the measurement was performed twice more in the same manner (third measurement set). Dust is not a special thing, but the things that are indoors are collected and thrown in everyday.

In FIG. 5, α is an offset value, A is a measured value for the reagent solution a, and B is a measured value for the reagent solution b. The value obtained by subtracting the offset value from each measured value and the finally obtained fluorescence intensity ratio are shown together.
In the combination of the liquid phase reagents a and b, since the concentration ratio is 5 times, the calculated fluorescence intensity ratio (known fluorescence intensity ratio in this example) is 5 times. Since the same combination of liquid phase reagents a and b is used in each measurement set, the measurement result (fluorescence intensity ratio) must be about 5 times as well, but as shown in FIG. Is fluctuating unstable. Since each measurement is performed continuously in time, it is difficult to consider fluctuation factors other than the intentional introduction of dust, such as fluctuations in the output of the light source 2. Even if an output fluctuation or the like of the light source 2 occurs between the measurement intervals of each set, since the ratio is taken, there should be no influence. Further, since the amount of dust intrusion in the optical system 4 is different, the background noise fluctuates. However, since it is measured in advance as an offset value and subtracted from the measured value, the influence should be removed, but the fluorescence intensity ratio is It has fluctuated.

What is interesting is that the offset value increases when dust is added and the amount is increased. The offset value should not fluctuate if the thrown-in dust only acts to shield excitation light and fluorescence. The fact that the offset value rises due to the introduction of dust is considered to directly indicate that the entered dust acts as a fluorescence generation source.
Further, when the first measurement set and the second measurement set are compared, the offset values of the measurement values A and B (the offset subtraction is performed although the second measurement set is larger by about 15.5 mV). The previous measurement value) is smaller in the second measurement set. It is considered that this is because dust that has entered the optical system 4 acts as a fluorescence generation source, but causes many effects of shielding the excitation light and the fluorescence from the reagent.
On the other hand, in the third measurement set, the offset value further increases by about 29.0 mV, and the measurement values A and B are conversely larger than the second measurement set and larger than the first measurement set. This result shows that as a result of a lot of dust adhering to the elements of the optical system 4, the action of the dust itself as a fluorescence generation source is stronger than the action of shielding the fluorescence from the excitation light and the reagent. It is thought that there is.

  In this way, it is assumed that foreign substances such as dust are mixed in the process of continuing use, and the measured fluorescence intensity values are different. When the dissociation of the measured value affects the value of the fluorescence intensity ratio and a correct determination cannot be made, it becomes a fatal problem as a fluorometer. Therefore, as described above, an inspection container (hereinafter referred to as an inspection reagent container) containing two types of liquid phase reagents as a standard having a known ratio of the fluorescence intensity generated is prepared. Fluorescence measurement is performed twice using a reagent container, and the accuracy of the fluorometer can withstand practical use by checking how much the ratio of the measured values deviates from the standard value. It is conceivable to judge whether or not. The inventor of the present application has invented the inspection method, the reagent container for inspection, and the like of the following embodiment based on such knowledge and examination.

  FIG. 6 is a schematic view of an inspection reagent container according to an embodiment of the present invention. The inspection reagent container 9 shown in FIG. 6 is substantially the same as the sample container 8 shown in FIG. Therefore, the inspection reagent container 9 can arrange the first inspection liquid phase reagent and the second inspection liquid phase reagent at the measurement position. In this embodiment, the second inspection liquid phase reagent is obtained by mixing another material with the first inspection liquid phase reagent. Specifically, as in the experiment described above, the first inspection liquid phase reagent and the second inspection liquid phase reagent are the same fluorescent dye, and are dissolved in the solution at different concentrations. Yes. The first container 91 of the inspection reagent container 9 stores the first inspection liquid phase reagent 93 in advance, and the second container 92 stores another material 95. Another material 95 is mixed with the first liquid reagent for inspection in the first container 91 by breaking the partition wall 94.

  In the inspection reagent container 9 of the embodiment, the ratio of the generated fluorescence in the first and second two liquid phase reagents for inspection is known, and the ratio (hereinafter referred to as the standard fluorescence intensity ratio). ) Information can be provided to the fluorometer 1. In addition, the inspection reagent container 9 according to the embodiment makes the fluorometer 1 unusable when the fluorescence intensity ratio obtained by using the inspection reagent container 9 deviates from the standard fluorescence ratio. Information on whether or not a reference value (hereinafter, referred to as “availability determination value”) can also be provided to the fluorometer 1.

More specifically, the inspection reagent container 9 according to the embodiment is configured to encode the standard fluorescence intensity ratio and the information on the acceptability determination value and provide them to the fluorometer 1. On the other hand, the fluorometer 1 includes a code reader that reads encoded information.
More specifically, as shown in FIG. 6, the inspection reagent container 9 of the embodiment has a portion (hereinafter referred to as a code display portion) 96 in which the standard fluorescence intensity ratio and the possibility determination value are displayed as barcodes. ing. The inspection reagent container 9 has a flat surface portion 97 on the side surface. In this embodiment, the code display unit 96 is formed by printing a barcode on a sticker and pasting it on the flat surface part 97.

On the other hand, the fluorometer 1 includes a barcode reader 7 as a code reader. As shown in FIG. 2, the barcode reader 7 is attached to the container mounting portion 5. The container mounting portion 5 has a reading opening 51 at a midway height. A reader mounting portion 52 is formed so as to protrude obliquely upward from the edge of the reading opening 51. The reader attachment part 52 is a short cylindrical part. The bar code reader 7 is fitted and held in the reader mounting portion 52 and can irradiate light or receive light through the reading opening 51.
As shown in FIG. 3, the reading opening 51 and the reader mounting portion 52 are formed on the side surface on the center side. “Center side” means the center side of the fluorometer 1. Therefore, the barcode reader 7 also reads from the center side.

FIG. 7 is a diagram showing a schematic configuration and an operation principle of the barcode reader 7 shown in FIG. (1) in FIG. 7 shows a state in which the barcode reader 7 is reading the code display unit 96, and (2) shows a state in which the barcode reader 7 is operating as a sensor for detecting the mounting of the sample container 8. As shown in FIG. 7, the barcode reader 7 includes a light emitter 71 that emits light to the code display unit 96, a light receiver 72 that receives light from the code display unit 96, and an output signal from the light receiver 72. And a binarization element 73 for processing to obtain a digital signal.
The inspection reagent container 9 is inserted from the insertion hole 50 at the upper end of the container holding part 5 shown in FIG. As shown in FIG. 1, the insertion hole 50 has a shape having a linear portion, and the flat surface portion 97 of the inspection reagent container 9 is aligned with the linear portion of the insertion hole 50 at the time of insertion. 97 will face the center side. When the inspection reagent container 9 is pushed down in this state, as shown in FIG. 2, the code display unit 96 passes in front of the reading opening 51, and at this time, the code display unit 96 is moved by the barcode reader 7. Read.

  More specifically, in this embodiment, the position of the barcode reader 7 is fixed, and the code display unit 96 moves relative to this position. The light emitter 71 irradiates a certain area with light, and the code display unit 96 passes through the light irradiation area. Accordingly, the code display unit 96 is irradiated with light when the lower end portion reaches the light irradiation region, and the upper portion is sequentially irradiated with light as it moves. Then, when the movement is completed (when the attachment is completed), the light irradiation has been completed up to the upper end portion. That is, the light irradiation region is relatively scanned by the movement of the inspection reagent container 9.

As shown in FIG. 7A, during scanning, light from the code display unit 96 irradiated with light enters the light receiver 72, and the binarization element 73 processes the output. The light that enters the light receiver 72 reflects the brightness of the barcode, and the binarization element 73 outputs a digital signal accordingly. As shown in FIG. 3, the barcode reader 7 of this embodiment irradiates light from an oblique direction, but light is diffused or scattered on the surface of the code display unit 96, and the light is captured by the light receiver 72. It will be. In many cases, it is recommended that the light receiving device 72 does not receive strong reflected light from the light emitting device 71. In this embodiment, the barcode reader 7 is attached obliquely in consideration of this.
As such a barcode reader 7, for example, RPU813T manufactured by Okaya Electric Industry Co., Ltd. can be used. Moreover, what used the laser for the light-emitting device 71 may be used, for example, H0A6480 etc. of Honeywell company can be used.

FIG. 8 is a schematic diagram showing an example of the format of the code display unit 96 shown in FIG. As shown in FIG. 8, in this example, 11-digit numbers are converted into barcodes. Of these, the first 1 digit is a type ID, the next 6 digits are expiration date information, the next 2 digits are standard fluorescence intensity ratios, and the next 2 digits are availability determination values.
The type ID is a code for causing the photometer to confirm that the attached container is the inspection reagent container 9. For example, when any number from 0 to 8 is given as information for specifying the sample container 8 attached during normal measurement, “9” is given as an ID indicating that it is the inspection reagent container 9. .

  The expiration date information indicates the expiration date of the inspection reagent solution. In many fluorescent dyes, a period during which a predetermined fluorescence characteristic is exhibited is limited to a certain period after manufacture, and thus expiration date information is included in the encoded information. In this embodiment, in the expiration date information, the first two digits are abbreviations of the Western calendar (13 in 2013), the next two digits are the month, and the next two digits are the day. . If it is 131020, it means that October 20, 2013 is the expiration date.

In this embodiment, the standard fluorescence intensity ratio is such that the first digit is the first digit and the next digit is the first digit after the decimal point. Therefore, “45” means that the standard fluorescence intensity ratio is 4.5 times.
In addition, the first and second digits are also in the first place, and the next one digit is in the first place after the decimal point. Therefore, if “05”, 0.5 is a determination value, and the difference between the fluorescence intensity ratio actually measured using the inspection reagent container 9 and the standard fluorescence intensity ratio is 0.5. If it is within the range, the fluorometer is determined to be usable.
The above is an example, and the standard fluorescence intensity ratio may be one digit or three digits or more. In addition, the possibility determination value may be a numerical value with two decimal places by giving three digits.

  On the other hand, the program stored in the memory 62 of the fluorometer 1 includes a program for inspection using the inspection reagent container 9 (hereinafter referred to as an inspection program). The menu screen displayed on the display 13 includes a menu item such as “measurement accuracy check”. This menu item uses the measurement button 141 as a check program start button, and when the “measurement accuracy check” is displayed, pressing the measurement button 141 starts the check program.

  FIG. 9 is a flowchart showing an outline of the inspection program. As shown in FIG. 9, the inspection program displays on the display 13 a screen that prompts the user to install the inspection reagent container 9, and waits to determine whether or not the inspection reagent container 9 has been installed. That is, the fluorometer includes a container sensor (not shown). The inspection program confirms whether or not the container is mounted by the output from the container sensor, and when the completion of the container mounting is confirmed, the output signal of the barcode reader 7 is processed (decoded), and the first one-digit information ( Type ID). When the type ID is information indicating that the inspection reagent container 9 is present, it is confirmed that the inspection reagent container 9 has been mounted. Therefore, the inspection program reads the standard fluorescence intensity ratio from the output of the barcode reader 7. Are taken out and temporarily stored in memory variables.

Next, the inspection program displays on the display 13 a message confirming the mounting of the inspection reagent container 9, and displays a message on the display 13 to perform the first measurement for inspection. When the measurement button 141 is pressed, the inspection program turns on the light source 2 and temporarily stores the measurement value output from the detector 3 in a memory variable.
Next, the inspection program displays a message on the display 13 to perform the second measurement for inspection. That is, the partition wall 94 of the inspection reagent container 9 is broken, and another material 95 in the second storage portion 92 is charged into the first storage portion 91, so that the second inspection liquid phase reagent is stored in the first storage portion 91. A message prompting the user to press the measurement button 141 in a state of being accommodated in the unit 91 is displayed on the display 13. When the measurement button 141 is pressed, the inspection program turns on the light source 2 again, and temporarily stores the measurement value output from the detector 3 in another memory variable.

Next, the inspection program reads the measurement values from each memory variable and calculates the ratio (the magnitude of the second measurement value with respect to the first measurement value). Then, the calculated ratio is compared with the standard fluorescence intensity ratio stored in the memory variable, and the difference is calculated. Then, the calculated difference is compared with the availability determination value stored in the memory variable, and it is determined whether the difference is equal to or less than the availability determination value. Although the difference may be negative, in this embodiment, it is determined by an absolute value whether or not the difference is equal to or less than the determination value. If it is equal to or less than the determination value, a message indicating that the fluorometer is normal and can be used is displayed on the display 13. If the determination value is exceeded, the fluorometer displays on the display 13 a message indicating that the measurement accuracy has deteriorated beyond the limit and is unavailable. This completes the inspection program.
Although illustration and description are omitted, the inspection program takes out the expiration date information from the output of the barcode reader 7 and determines whether the expiration date has passed. If the expiration date has passed, a message to that effect is displayed on the display 13, and the program is terminated without performing an inspection operation (two measurements).

Based on the above description, an embodiment of the fluorometer inspection method will be described below. It is recommended that the fluorometer 1 as described above is periodically checked for measurement accuracy by an operation manual or the like. In addition, an inspection kit including the above-described inspection reagent container 9 is provided separately from the fluorometer 1. The inspection kit is obtained by enclosing an inspection reagent container 9 in an individual bag.
As described above, the menu item of “measurement accuracy check” is displayed on the display 13, and in this state, the measurement button 141 is pressed to start the inspection program. First, since the installation of the inspection reagent container 9 is urged, the inspection reagent container 9 is taken out from the individual packaging bag, and the opening / closing lid 12 is opened and inserted into the casing 10 through the insertion hole 11 as in the case of normal measurement. To do. The inspection reagent container 9 is mounted on the container mounting portion 5 in the same manner as the normal sample container 8, and the first container 91 is located at the measurement position.

Next, the measurement button 141 is pressed according to the display on the display 13, the light source 2 is turned on, and measurement (first measurement step) is performed. Next, according to the display on the display 13, the partition wall 94 in the inspection reagent container 9 is broken, and another material in the second storage portion 92 is replaced with the first inspection liquid phase reagent 93 in the first storage portion 91. To mix. Thereby, the inside of the 1st accommodating part 91 will be in the state in which the 2nd liquid reagent for inspection was accommodated. In this state, the measurement button 141 is pressed again to turn on the light source 2 and perform measurement (second measurement step).
Then, the inspection program causes the arithmetic processing unit 61 to perform the arithmetic processing, and the comparison result with the determination value is displayed on the display 13. If the result is normal, the inspection reagent container 9 is taken out, a normal sample container 8 is mounted, and the original measurement is performed. If the result is abnormal, the display is disabled, so stop the measurement and use another fluorometer.

According to the inspection method of the embodiment, two liquid phase reagents with known fluorescence intensity ratios are put into a fluorometer to be inspected and measurement is performed to obtain a ratio of measured values. It can be checked how far is dissociated from the standard fluorescence ratio. For this reason, it is possible to prevent an erroneous measurement result from being obtained by performing measurement without knowing the decrease in measurement accuracy.
In addition, it is possible to set the acceptability judgment value in consideration of the case where foreign matter is mixed in the fluorometer and the foreign matter is a fluorescent substance, so the fluorometer used in an environment or situation where foreign matter is likely to be mixed This is a particularly suitable inspection method. For example, in the case of a portable fluorometer as described above, measurement is possible in various places, but it is also assumed that the portable fluorometer is used in an environment or situation in which foreign matters such as dust and dust are easily mixed. Therefore, it can be said that the inspection method of the embodiment is a method particularly suitable for a portable fluorometer.

In addition, an actual measurement is performed on two liquid phase reagents with known fluorescence intensity ratios, and an inspection is performed by obtaining the ratio, and a fluorometer that obtains the ratio of fluorescence intensities of two samples as a measurement result In addition to being optimized as a target inspection method, there is a significance that the influence of background noise can be easily checked while canceling factors such as light output fluctuation and light shielding due to contamination of the optical system.
In other words, when checking the measurement accuracy of a fluorometer, the measurement is performed for a standard liquid phase reagent whose absolute value of the generated fluorescence is known, and the absolute value of the luminance calculated from the output obtained by the detector. Is first considered to be compared with the known absolute value of luminous intensity. Then, when the dissociation of the two absolute values exceeds a certain value, the fluorometer can be disabled.

  Such an inspection method may be used, but the difference between the two absolute values (noise) includes factors such as light shielding due to deterioration of the light source and adhesion of foreign substances in the optical system in addition to background noise. The noise factor is not a problem when taking a ratio of measured values in actual measurement. In other words, with an inspection method that compares two absolute values, noise that is not actually a problem will be included in the judgment element, and even a precision decrease that does not actually cause a problem is a problem. It may be judged that. For example, even if there is only a decrease in accuracy due to fluctuations in the output of the light source and there is no increase in background noise due to factors such as contamination of foreign substances that are fluorescent substances, the two are not distinguished, so they are used due to a decrease in measurement accuracy It will be the inspection result that it is impossible. According to the method of the embodiment, there is no such problem, and it is possible to perform an inspection for checking only the problem of accuracy degradation.

  Further, in the method of the embodiment, after the first measurement step is performed in a state where the first container 91 containing the first inspection liquid phase reagent 93 is disposed at the measurement position, another material 95 is added By mixing with one inspection liquid phase reagent 93, the second inspection liquid phase reagent is positioned at the measurement position, and the second measurement step is performed in this state. That is, the second measurement step can be performed subsequent to the first measurement step without changing the position or posture of the inspection reagent container 9. For this reason, an inspection without error can be easily performed.

To implement the method of the present invention, a first inspection reagent container containing a first inspection liquid phase reagent and a second inspection reagent container containing a second inspection liquid phase reagent are provided. It can also be used. In this case, since different containers are used, a difference in conditions between the containers may affect the measurement. Further, when the container is replaced, foreign matter such as dust may enter from the insertion hole, and the measurement conditions may also change in this respect. In addition, the inspection liquid phase reagent storage unit is provided at the top and bottom of one inspection reagent container, and the first and second inspection liquid phase reagents are sequentially placed at the measurement position by mounting them upside down. The structure can be considered, but in this case as well, since the container wall portion of the container is different in the first and second liquid reagents for inspection, the measurement conditions change, such as the presence or absence of foreign matter adhering to that portion. There is a possibility that. If the measurement conditions change, the ratio of the measured values may not match the actual fluorescence intensity ratio, which may affect the accuracy of the determination of whether or not the fluorescence intensity meter can be used. Absent.
The structure in which another solution 95 in the second container 92 is moved to the first container 91 and mixed with the first liquid phase reagent 93 is the second breakable partition wall 94 described above. In addition to the structure that forms the bottom of the housing portion 92, a structure in which the bottom portion of the second housing portion 92 is formed by a slider that can be operated from the outside can be employed.

  Also, the fact that the first inspection liquid phase reagent and the second inspection liquid phase reagent are the same reagent and have different concentrations also has the significance of facilitating inspection without error. Any one of the first inspection liquid phase reagent and the second inspection liquid phase reagent can be used as long as the fluorescence intensity ratio is known. However, when different reagents are used, there is a possibility that variations in the manufacture of the reagents will be affected. When the same reagent is used and the concentration is different to make the first inspection liquid phase reagent and the second inspection liquid phase reagent, the reagents are the same. And manufacturing variations can be canceled. Even if the fluorescence intensity (absolute value) of a reagent in a certain production lot deviates from the specified value, the first and second liquid phase reagents are prepared with the same production lot reagent, and the intensity ratio of the fluorescence generated from them. As long as inspection is carried out at, deviation of the absolute value from the specification value does not matter.

In addition, since the inspection reagent container 9 of the embodiment has the code display unit 96 displaying the standard fluorescence ratio, the user inspected the fluorometer using the inspection reagent container 9. In this case, it can be compared with the measured fluorescence intensity ratio. Therefore, it can be easily determined whether or not the fluorometer can be used.
Other means for informing the user of the standard fluorescence intensity ratio include printing on a label and sticking it on the individual packaging of the reagent kit for inspection, or enclosing a printed piece of paper in the individual packaging. . In addition, if the standard fluorescence ratio is a common and constant value, there is also a method of describing it in the spectrophotometer specifications or instruction manual. Nonetheless, when displayed on the reagent container 9, it is possible to check even if an individual bag or a piece of paper is lost or discarded, and it is possible to cope with a case where the value is different for each reagent container 9. Is possible.

  The configuration in which the fluorometer 1 includes the barcode reader 7 and the code display unit 96 of the reagent container 9 is a code display unit 96 that can be read by the barcode reader 7 is based on the standard fluorescence ratio and the possibility determination value. Is suitable for causing the fluorometer 1 to acquire the above. In order for the fluorometer 1 to automatically determine whether or not it is possible from the measurement results of the two liquid phase reagents for inspection and to inform the user of the result, it is necessary to acquire the standard fluorescence ratio and the determination value as digital information. . For this purpose, there may be a method in which the user reads the display unit and inputs it manually into the fluorometer, but it is troublesome. If the bar code reader 7 reads the code display unit 96 as in the embodiment, there is no troublesomeness. Moreover, in the structure of the embodiment, since the code display unit 96 is automatically read in the operation of mounting the inspection reagent container 9 on the container mounting unit 5, this is also a very simple method. .

Note that the fact that the code display unit 96 displays the availability determination value in addition to the standard fluorescence ratio has the significance that the availability determination of the fluorometer 1 can be easily performed without error. Whether or not the standard fluorescence intensity ratio alone is used can be determined to some extent by comparing it with the measured fluorescence intensity ratio. For example, it is possible to determine whether or not the difference between the standard fluorescence intensity ratio and the measured fluorescence intensity ratio is within a tolerance range that the user knows empirically. However, since this method relies on the user's experience, there is a problem that the judgment is not reliable. In addition, when the deviation tolerance is a common general value, there is a method to describe it in the spectrophotometer specifications and instruction manual, but you should refer to the specifications and instruction manual one by one. It must be cumbersome. According to the reagent container for inspection 9 of the embodiment, there is no such problem.
In addition, the fluorometer 1 includes an arithmetic processing unit 61 and an inspection program, and an operation for obtaining a fluorescence intensity ratio from a measurement value, an operation for obtaining a difference by comparing the obtained fluorescence intensity ratio with a standard fluorescence intensity ratio, and the like. What is performed by the arithmetic processing unit 61 does not require the user to perform such a calculation, and thus becomes a highly convenient fluorometer in this respect.

DESCRIPTION OF SYMBOLS 1 Fluorometer 10 Casing 2 Light source 3 Detector 4 Optical system 5 Container mounting part 6 Control part 7 Bar code reader 8 Sample container 9 Reagent container 91 First container 92 Second container 93 First inspection Liquid phase reagent 94 Septum 95 Other material 96 Code display part

Claims (9)

Place the sample container containing the liquid phase sample in the container so that the container is at the measurement position, and irradiate the measurement position with excitation light and capture the fluorescence generated in the liquid sample with the detector. An inspection method,
It is an inspection method using an inspection reagent container in which the first and second two liquid phase reagents for inspection whose fluorescence intensity ratio is known can be sequentially arranged at the measurement position,
The first measurement in which the detection light is captured by the detector by irradiating the measurement light with the excitation light from the light source in a state where the storage portion of the inspection reagent container containing the first inspection liquid phase reagent is positioned at the measurement position Steps,
Second measurement in which the measurement light is irradiated with the excitation light from the light source and the generated fluorescence is captured by the detector in a state where the container of the inspection reagent container containing the second inspection liquid phase reagent is positioned at the measurement position. Steps,
The ratio of the fluorescence intensity captured by the detector in the first measurement step and the fluorescence intensity captured by the detector in the second measurement step is obtained, and the obtained ratio and the known fluorescence intensity And a calculation step of calculating a difference from the ratio. The second inspection liquid phase reagent is formed by mixing another material with the first inspection liquid phase reagent,
After the first measurement step, another material is mixed with the first inspection liquid phase reagent stored in the storage unit, and the second inspection liquid phase reagent is mixed in the storage unit. 2. The method of checking a fluorometer according to claim 1, wherein the second measuring step is performed after the container is housed. 3. The fluorometer inspection method according to claim 2, wherein the first inspection liquid phase reagent and the different material are prepared by dissolving the same fluorescent reagent in a solution at different concentrations.   The reagent container for use in the method for inspecting a fluorometer according to claim 1, 2, or 3, comprising a display unit displaying the known fluorescence intensity ratio. container.   The display unit displays a feasibility judgment value in addition to the known fluorescence intensity ratio, and the feasibility judgment value indicates a magnitude of a difference between the obtained ratio and the known fluorescence intensity ratio. The inspection reagent container according to claim 4, wherein the inspection reagent container is a reference value that is a value that disables the use of the fluorometer when the difference exceeds a determination value.   6. The inspection reagent container according to claim 4, wherein the display unit is a code display unit that can be read by a code reader included in the fluorometer.   A reagent container for inspection used in the method for inspecting a fluorometer according to claim 2, wherein a first storage part storing the first inspection liquid phase reagent and a first storage part storing the another material are provided. And having a structure capable of being mixed with the first liquid phase reagent by moving the other material from the second storage part to the first storage part. Characteristic reagent container for inspection. A fluorometer in which the fluorometer inspection method according to claim 1, 2, or 3 is implemented,
A storage unit, an arithmetic processing unit, and a display;
The storage unit measures the fluorescence intensity of the first inspection liquid phase reagent measured in the first measurement step, and the second inspection liquid phase reagent measured in the second measurement step. The measured value of the fluorescence intensity can be stored, the inspection program is stored in the storage unit and can be executed by the arithmetic processing unit,
The inspection program is a program that obtains a ratio between the fluorescence intensity of the first inspection liquid phase reagent and the fluorescence intensity of the second inspection liquid phase reagent and displays the ratio on a display. . A fluorometer in which the fluorometer inspection method according to claim 1, 2, or 3 is implemented,
A storage unit, an arithmetic processing unit, and a display;
The storage unit measures the fluorescence intensity of the first inspection liquid phase reagent measured in the first measurement step, and the second inspection liquid phase reagent measured in the second measurement step. The measured value of the fluorescence intensity of
An inspection program is stored in the storage unit and can be executed by the arithmetic processing unit.
The inspection program obtains the ratio between the fluorescence intensity of the first inspection liquid phase reagent and the fluorescence intensity of the second inspection liquid phase reagent, and then whether or not the obtained difference exceeds the determination value. A fluorometer that is a program that, when judged and exceeded, displays on the display that the fluorometer is unusable.

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